JP2017067498A - Method for capturing or detecting circulation cancer cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for simply capturing and detecting a cancer cell such as a neuroblastoma in a non-invasive manner.SOLUTION: A method for capturing a circular cancer cell includes a process of capturing a circular cancer cell contained in a liquid sample in a microchip by allowing the liquid sample derived from the blood isolated from a subject to pass through the microchip in vitro. The microchip has a flow channel capable of allowing the fluid to pass through the interior, an inflow port for allowing the liquid to flow into the flow channel, a discharge port for discharging the liquid having passed the flow channel, and an antibody for capturing the circular cancel cell carried on the flow channel. The process immerses the microchip, and introduces a sample into the microchip from the inflow port in a state that the discharge port side is immersed into water and the inflow port side is placed above the water surface.SELECTED DRAWING: Figure 2

Description

The present invention relates to a method for capturing or detecting circulating cancer cells present in a sample derived from a subject using a microchip having an antibody immobilized therein.

In the diagnosis of cancer, it is difficult to directly analyze cancer tissues and organs at an early stage where cancer is suspected, and there is a need for a simple and noninvasive technique for early examination. On the other hand, cancer cells may leak into the blood from cancer tissue, and if cancer cells circulating in such blood can be detected, simple diagnosis of cancer becomes possible.
As a device for detecting circulating cancer cells (CTC), a diagnostic device using a circulating cancer cell (CTC) chip is known (Patent Document 1). The CTC chip is a microchip carrying a tumor cell-specific antibody inside, and circulating cancer cells can be captured and evaluated by flowing a blood sample through the flow path. However, the conventional method requires a precise syringe pump to flow a blood sample, and requires precise and precise liquid feeding (injection and aspiration). In addition, cancer cells and blood cells in a specimen are required. However, there is a problem such as the need for a device that continuously shakes and holds the sample so as not to form an agglomerate, and no actual clinical application has been made.

JP 2013-029391

An object of the present invention is to provide a method for capturing and detecting cancer cells such as neuroblastoma in a simple and non-invasive manner.

As a result of intensive studies to solve the above problems, the present inventor has obtained a sample derived from the peripheral blood of a subject as a microchip carrying an antibody for capturing cancer cells (hereinafter also referred to as a CTC chip). The sample is introduced into the microchip from the inlet and allowed to react with the antibody while the outlet side of the microchip is immersed in water and the inlet side is placed above the water surface. It has been found that circulating cancer cells can be detected by simple examination, and the present invention has been completed.

That is, the present invention is a method of capturing circulating cancer cells,
In vitro, passing a blood-derived liquid sample isolated from a subject through the microchip and capturing the circulating cancer cells contained in the liquid sample in the microchip,
The microchip has a flow path through which a fluid can pass, an inlet for allowing liquid to flow into the flow path, an outlet for discharging the liquid that has passed through the flow path, and a support on the flow path. Having an antibody for capturing the circulated cancer cells,
The step is performed by introducing the sample into the microchip from the inlet while the microchip is immersed in water on the outlet side and the inlet side is placed above the water surface. To do.

According to the method of the present invention, it becomes possible to capture CTCs of patients with intractable solid tumors such as neuroblastoma in a simple, inexpensive and short time and minimally invasive manner. That is, the present invention has the following advantages.
1) It is easy to prepare for inspection as an inspection device.
2) Do not use unnecessary reagents because there is no need to use tubes.
3) Since there is no useless channel other than the microchip, the number of samples can be reduced, leading to a reduction in examination time and a burden on the patient.

The figure which shows the one aspect | mode of a CTC chip. (A) is the first substrate, (B) is the second substrate, and (C) is a completed drawing. The figure which shows the example of the usage condition of a CTC chip. The microscope picture which shows a result when a NB69 tumor cell is mixed with a peripheral blood mononuclear cell (PBMC) sample, and it used for the CTC chip | tip. The microscope picture which shows a result when PBMC derived from a neuroblastoma patient is used for a CTC chip. The micrograph which shows the result of having dye | stained with PLS3 antibody or DAPI, after using PBMC derived from a neuroblastoma patient for a CTC chip.

In the present invention, the circulating cancer cells to be captured and detected are not particularly limited as long as they are cancer cells that can circulate in blood, but human cancer cells are preferable, and neuroblastoma, lung cancer, esophageal cancer, gastric cancer And refractory solid tumors such as GIST, colorectal cancer, pancreatic cancer and breast cancer.

As a sample containing circulating cancer cells, a sample derived from the blood of a subject can be used, but peripheral blood mononuclear cells (PBMC) are preferably used. The amount of the sample used for one measurement is not particularly limited, but according to the method of the present invention, a small amount of sample of about several tens to several hundreds μL (specifically, for example, 50 to 300 μL) is used. Measurement is possible.

In the present invention, the microchip used has a flow path through which fluid can be passed and an antibody for capturing circulating cancer cells carried on the flow path. What is necessary is just to have the inflow port for making a liquid flow in into a flow path in a 1st end, and the discharge port for discharging the liquid which passed the flow path in the 2nd end of a flow path. For example, those disclosed in JP 2013-029391 A can be used.

FIG. 1 shows one embodiment of a microchip.
Although (C) is a completed drawing, such a microchip can be manufactured as follows, for example.
First, a first substrate 101 in which a groove 102 to be a flow path is dug on one side is prepared, and a plurality of columnar members 103 serving as carriers for immobilizing antibodies are arranged in the flow path portion. A functional group is modified on the surface of the columnar member 103, and the antibody can be immobilized via the functional group. The antibody may be immobilized in advance, or may be immobilized by passing the antibody solution through the channel immediately before use. Note that the member for immobilizing the antibody is not limited to a columnar shape, and the antibody may be directly immobilized on the flow path. The size of the flow path is not particularly limited as long as it is a size suitable for flowing a sample containing circulating cancer cells. For example, the width can be 10 to 50 mm, the length can be 30 to 100 mm, and the depth can be 50 to 200 μm. For example, a plurality of columnar members having a diameter of 50 to 200 μm and a height of 50 to 200 μm can be installed in the flow path at an appropriate interval (a level that does not hinder the flow of a sample containing circulating cancer cells). The volume of the portion that can be filled with the liquid in the flow path is preferably 30 to 200 μL.

Next, a second substrate 104 having two holes (through-holes) to be the inlet 105 and the outlet 106, respectively, is prepared, and this is overlapped with the first substrate 101 so that the groove 102 is inside. Thus, the microchip 100 (CTC chip) can be manufactured.
The material of the microchip is preferably a material that does not cause nonspecific adsorption of cells and is transparent and the inside can be observed. Examples thereof include materials such as polystyrene, polyethylene, polypropylene, and PET, and glass.

The antibody for capturing circulating cancer cells is preferably an antibody against a marker protein specific to cancer cells, and is appropriately selected depending on the type of circulating cancer cells of interest, but it is an antibody against EpCAM, an epithelial cell marker. , An antibody against CD44, a surface marker, an antibody against CD133, an antibody against cell surface receptor EGFR, an antibody against ERBB2, an antibody against ERBB3, an antibody against solid tissue marker PLS3, etc. In some cases, it is preferable to use an anti-CD47 antibody. CD47 is a transmembrane protein also known as integerrin associated protein.
The antibody may be a polyclonal antibody or a monoclonal antibody. A commercially available antibody can be used as the antibody, but the antibody may be prepared and used by a hybridoma method or the like. Two or more antibodies may be immobilized on the microchip.

For example, the sample can be added to the microchip as follows.
As shown in FIG. 2, first, a container containing water is prepared, and the microchip outlet side is immersed in water (the inlet side is not immersed in water, but is placed in the atmosphere above the water surface). Tilt the tip and place it. The installation angle of the microchip with respect to the water surface is, for example, 25 to 60 degrees. The water in the container may be physiological saline or an aqueous buffer solution.

And a sample is introduce | transduced into the flow path in a microchip from an inflow port. Examples of the introduction method include a method of dropping with a pipette or the like, but are not particularly limited.
By placing the microchip's outlet side in water, the sample and reagent containing cells flow at an almost constant flow rate based on the surface tension (capillary phenomenon) of the specimen itself and the pressure difference between the inlet and outlet. Is possible, so that CTC in the sample can be stably captured.

After introducing the sample into the microchip and passing through the channel, it is preferable to perform a washing operation in order to remove non-specific binding of cells and the like. The washing operation is preferably performed once or more using, for example, a buffer solution such as a phosphate buffer.

Detection of the captured cells may be performed visually using a microscope or the like, but may also be performed using a cell staining reagent or a cancer cell-specific antibody. In that case, a detection reaction may be performed by introducing a reagent or an antibody reaction solution from the inlet.

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, the content of this invention is not limited to the following.

First, CTC chips were obtained from the Toyama Industrial Technology Center. The size is as follows.
76mm length
26mm wide
Length of channel (groove): 40mm, width: 19mm, depth: 100μm
Columnar size: radius 50μm, height 100μm
Functional group type: Epoxy base inlet diameter 1mm, outlet diameter 1mm

PBS buffer was passed through the CTC chip to equilibrate the flow path of the CTC chip.
Next, anti-CD47 antibody (mouse anti-CD47 monoclonal antibody: Santa Cruz Biotechnology) was diluted 100 times with PBS to a concentration of 20 μg / mL, introduced into the flow path, and the columnar member in the CTC chip. It was immobilized by reacting with a functional group on the surface. After reacting for 1 hour at room temperature, the antibody solution was washed with PBS. On the other hand, as a control, an anti-mouse IgG antibody (Southern biotech) was immobilized in a CTC chip at 20 μg / mL (4 ° C. overnight or room temperature for 3 hours).

First, 250 μL of PBMC obtained from healthy human blood was prepared, and 2000 human neuroblastoma cell line NB69 (RIKEN cell bank) was added thereto as a measurement sample.
As shown in Figure 2, prepare a container with water, soak the CTC tip outlet side in water, hold the inlet side so that it is located above the water surface, and in that state, use a pipette. The measurement sample was dropped into the inlet (each 10 μL) and introduced into the channel of the CTC chip.
After washing with 400 μL of PBS, the inside was observed with a microscope. The results are shown in FIG.
As shown in FIG. 3, cell adhesion was observed around the carrier (columnar member) on which the anti-CD47 antibody was immobilized, and it was confirmed that the cancer cells could be captured by the anti-CD47 antibody. Moreover, the capture rate of cancer cells was about 30%.

Next, 100 μL of PBMC obtained from the blood of a child with neuroblastoma was prepared and used as a measurement sample.
As shown in Figure 2, prepare a container with water, soak the CTC tip outlet side in water, hold the inlet side so that it is located above the water surface, and in that state, use a pipette. The measurement sample was dropped into the inlet (each 10 μL) and introduced into the channel of the CTC chip.
After washing with 400 μL of PBS, the inside was observed with a microscope. The results are shown in FIG.
As shown in FIG. 4, cell adhesion is seen around the carrier (columnar member) on which the anti-CD47 antibody is immobilized, and the anti-CD47 antibody can capture cancer cells (CTC) contained in the patient's PBMC. It was confirmed that.

Furthermore, the cells captured in FIG. 4 were stained with an anti-PLS3 antibody (Santa Cruz Biotechnology) or DAPI to identify the cells. The staining procedure is as follows.
1. After washing blood cells with PBS 50μL x8 times, fix cells with 4% PFA 10μL x10 times at room temperature for 10 minutes
2. PBS 50μL x4 wash
3. Prepare PLS3 antibody (100-fold dilution: antibody 2 μL + PBS 198 μL) and transfer the entire amount to the chip. 20 minutes at room temperature
4. PBS 50μL x 4 washes
5. Prepare anti-goat 594 antibody (Molecular Probe) at 500-fold dilution (1 μL + 499 μL), and add 10 μL each to 200 μL to the CTC chip. Room temperature shading 20 minutes
6. PBS 50μL x 4 washes
7. Add 10 μL of nuclear staining solution DAPI solution to CTC chip.
8. Observation with a fluorescence microscope

The results are shown in FIG. As a result, the captured cells were positive for PLS3, which is a cancer cell marker, and it was confirmed that CTC was captured.

From the above, according to the method of the present invention, the method of placing the CTC chip outlet in water and flowing the sample or reagent into the CTC chip with the surface tension and pressure difference is achieved without using a syringe pump for a short time (about 90 minutes for the conventional method). (→ 10 minutes) CTC supplementation became possible. In addition, since no jigs are used, preparation is easy and no excessive circuit (tube) is required, so it is possible to save reagents, antibodies, and cells (about 1.5 mL → 50 μL of the conventional method).

DESCRIPTION OF SYMBOLS 100 ... Microchip (CTC chip), 101 ... First substrate, 102 ... Channel (groove), 103 ... Columnar member, 104 ... Second substrate, 105 ... Inlet, 106 ... discharge port, 107 ... container, 108 ... water

Claims (4)

A method for capturing circulating cancer cells, comprising:
In vitro, passing a blood-derived liquid sample isolated from a subject through the microchip and capturing the circulating cancer cells contained in the liquid sample in the microchip,
The microchip has a flow path through which a fluid can pass, an inlet for allowing liquid to flow into the flow path, an outlet for discharging the liquid that has passed through the flow path, and a support on the flow path. Having an antibody for capturing the circulated cancer cells,
The method is characterized in that the microchip is performed by immersing the sample into the microchip from the inlet while the microchip is immersed in water on the outlet side and the inlet side is placed above the water surface. The method according to claim 1, wherein the circulating cancer cell is a neuroblastoma-derived cancer cell. The method according to claim 1 or 2, wherein the antibody is an anti-CD47 antibody. Furthermore, the method as described in any one of Claims 1-3 including the process of detecting the captured circulating cancer cell.